Yersinia pestis and Francisella tularensis are the causative agents of plague and tularemia, respectively. Both of these bacteria are extremely virulent for humans when aerosolized and thus have the potential for use as agents of bioterrorism. Greater knowledge of the molecular mechanisms underlying Y. pestis and F. tularensis pathogenesis is urgently needed. The goals of this proposal are to characterize Y. pestis and F. tularensis virulence factors and to elucidate mechanisms of virulence factor biogenesis. Genome sequencing of Y. pestis revealed the presence of ten chaperone/usher secretion pathways, eight of which were previously unknown. Chaperone/usher pathways are utilized by a broad range of bacterial pathogens for the biogenesis of virulence-associated surface structures. The first specific aim of this proposal is to investigate the chaperone/usher pathways of Y. pestis. Expression of the novel pathways will be determined and the roles of the pathways in pathogenesis will be tested by aerosol infection of mice. Pathways with a virulence phenotype will be selected for further analysis. In addition, detailed analysis of F1 capsule biogenesis by the previously known caf chaperone/usher pathway will be performed. The F1 capsule is the major protective antigen of K pestis. The molecular basis for the virulence of F. tularensis is largely unknown. The second specific aim of this proposal is to identify and characterize potential virulence factors of F. tularensis, focusing on surface and secreted proteins. A bioinformatics approach will be used to mine the F. tularensis genome for secretion systems, secreted proteins and surface structures. Ultrastructural and biochemical methods will be used to directly visualize and isolate F. tularensis surface structures and secreted proteins. Potential virulence factors will be tested for their roles in pathogenesis by aerosol infection of mice. This proposal will be done in close collaboration with the other Projects and Core facilities of the Program Project Grant entitled "Agents of Bioterrorism: Pathogenesis and Host Defense." The work described in this proposal and the Program Project Grant will provide insights into the pathogenesis of Y. pestis and F. tularensis, and create opportunities for the development of novel methods to detect, prevent and treat outbreaks of plague or tularemia.